The present invention relates to stable crystals of p1,p4-di(uridine 5xe2x80x2-)tetraphosphate (U2P4) or a salt thereof which are useful as an active ingredient of an expectorant or a therapeutic agent for pneumonia; a process for producing the crystals; and a process for efficiently producing U2P4 or a salt thereof.
A tetrasodium salt of P1,P4-di(uridine 5xe2x80x2-) tetraphosphate (U2P4xc2x74Na) represented by the following formula (I): 
has an expectoration-inducing action and is a compound which is expected to be developed as an expectorant or a therapeutic agent for pneumonia (e.g., U.S. Pat. Nos. 5,789,391, 5,763,447, and 5,635,160).
Until now, U2P4 has not been obtained in crystal form, and has been prepared only in the form of a lyophilized product (see Example 1 of WO 99/05155). Typical U2P4 produced by conventional method has a purity as low as 90%, and contains by-products. Examples of by-products include nucleoside 5xe2x80x2-(poly)phosphates such as uridine 5xe2x80x2-tetraphosphate (UP4), uridine 5xe2x80x2-triphosphate (UTP), uridine 5xe2x80x2-diphosphate (UDP), and uridine 5xe2x80x2-monophosphate (UMP); and dinucleoside polyphosphates such as P1,P4-di(uridine 5xe2x80x2-) triphosphate (U2P3) and P1,P4-di(uridine 5xe2x80x2-)diphosphate (U2P2). Particularly, it is difficult to separate nucleoside 5xe2x80x2-(poly)phosphates such as UTP from U2P4, and highly purified U2P4 has been produced only with great difficulty through a conventional purification method; i.e., ion-exchange chromatography (WO 99/05155, Biochimica et Biophysica Acta, 438, (1976) 304-309).
The above purified and lyophilized product has disadvantages such as high hygroscopicity. Therefore, preparation of a pharmaceutical from U2P4 must be carried out in a special apparatus in which moisture is well controlled. Even after preparation of a pharmaceutical, the product must be wrapped tightly. In addition, since the pharmaceutical has a very short available period due to poor stability of the lyophilized preparation, obtaining highly purified and stable U2P4 crystals has been desired.
U2P4 is synthesized from uridine 5xe2x80x2-monophosphate (UMP) by use of an activating agent such as diphenyl phosphochloridate (DPC) and a phosphorylating agent such as a pyrophosphate (PPi). However, a conventional process provides a low synthesis yield; i.e., as low as approximately 10 wt. % (Example 4B of WO 99/05155), and can never serve as a practical process. Accordingly, development of a process for producing U2P4 at high yield and on a large scale has also been desired.
In view of the foregoing, an object of the present invention is to provide stable crystals of U2P4 or a salt thereof. Another object of the invention is to provide a process for producing the crystals. Still another object of the invention is to provide a process for efficiently producing U2P4 on a large scale.
The present inventors have conducted earnest studies on a method for purifying U2P4 and a process for synthesizing U2P4 from UMP. The inventors have found that U2P4 purified through anion exchange chromatography and chromatography using activated charcoal (activated-charcoal chromatography) can be easily crystallized and that use of specific reaction conditions has the effect of drastically increasing the yield of U2P4 in the synthesis of U2P4 or a salt thereof from UMP serving as a starting material and by use of DPC and PPi. The present invention has been achieved on the basis of these findings.
Accordingly, the present invention provides crystals of P1,P4-di(uridine 5xe2x80x2-)tetraphosphate or a salt thereof.
The present invention also provides a process for producing crystals of P1,P4-di(uridine 5xe2x80x2-)tetraphosphate or a salt thereof, which process comprises purifying crude P1,P4-di(uridine 5xe2x80x2-)tetraphosphate or a salt thereof through anion exchange chromatography and activated-charcoal chromatography and adding a hydrophilic organic solvent to a solution of purified P1,P4-di(uridine 5xe2x80x2-)tetraphosphate or a salt thereof, to thereby precipitate crystals.
The present invention also provides a process for producing P1,P4-di(uridine 5xe2x80x2-)tetraphosphate or a salt thereof from uridine 5xe2x80x2-monophosphate (UMP) serving as a starting material and by use of diphenyl phosphochloridate (DPC) and a pyrophosphate (PPi), which process comprises at least one of the following treatment steps:
(a) adding UMP diphenylphosphate (UMP-DPP) in divided portions during a step of reaction of UMP-DPP with a PPi-organic alkali salt;
(b) carrying out a step of reaction of UMP-DPP with a PPi-organic alkali salt in the presence of a base; and
(c) further treating the synthesized U2P4 with an alkali.